A mixture of nitrite-oxidizing and denitrifying microorganisms affects the δ18O of dissolved nitrate during anaerobic microbial denitrification depending on the δ18O of ambient water

The stable isotopes N-15/N-14 and O-18/O-16 of nitrate are frequently used to determine sources of nitrate and to assess denitrification processes in the environment. Nitrate isotope ratios are thought to be conservative unless involved in (bio-) chemical conversion processes. Thus, stable isotopes are considered to be a reliable tool to determine sources of nitrate in aquatic habitats even after transport and dilution has occurred. Denitrification is known to shift both isotope ratios towards higher delta-values. A fixed ratio of 0.5 for Delta delta O-18/Delta delta(15) N has been proposed and has been widely used to detect denitrification in terrestrial environments, predominantly in aquifers. However, it is observed in environmental and laboratory studies that this ratio actually varies between less than 0.5 and 1 for uncertain reasons with laboratory studies usually describing a ratio close to 1. Here we report results of anoxic incubation experiments with natural populations of nitrate-reducing microorganisms using sediments from three different environments. In our experiments we used water with a delta O-18 in excess of 500 parts per thousand and found a microbially mediated influence of the oxygen isotopic composition of ambient water on the isotopic composition of the residual dissolved nitrate. We found up to 5.7 +/- 2.3% of the oxygen-atoms in the residual dissolved nitrate was exchanged by oxygen-atoms from ambient water within the limited timeframe of the experiments. The fastest incorporation of oxygen-atoms from water into dissolved nitrate correlated with the highest intermittent nitrite concentrations observed in our experiments. In a second series of batch experiments we also found that pure cultures of the nitrite-oxidizing bacterium Nitrobacter vulgaris promoted the incorporation of oxygen atoms from ambient water into dissolved nitrate under anoxic conditions. Presumably this happens via a reoxidation of intermediary formed nitrite by the enzyme "nitrite oxidoreductase" (NXR) in concurrence with respiratory nitrate reduction. In this context, our hypothesis is a reversibility of the reactions at the NXR enzyme even in the absence of external electron acceptors for nitrite oxidation. We suggest that the presence of nitrite-oxidizing microorganisms in aquatic environments may catalyse such an incorporation of oxygen-atoms stemming from ambient water into nitrate. This process may thus mask the original delta O-18 value of nitrate sources during denitrification and also distort the observed enrichment of O-18 that is ascribed to denitrification. Our results are highly likely an explanation of the deviation of the described variable Delta delta O-18/Delta delta N-15 ratios for denitrification in terrestrial field studies from the values observed in the laboratory on pure cultures. (C) 2013 Elsevier Ltd. All rights reserved.